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Timothy P. Driscolla, Victoria I. Verhoevea, Mark L. Guillotteb Wholly Rickettsia! Metabolic Profile of the Quintessential Bacterial Parasite of Eukaryotic Cells Item Type Poster/Presentation Authors Driscoll, Timothy P.; Verhoeve, Victoria I.; Guillotte, Mark L.; Lehman, Stephanie S.; Rennoll, Sherri A.; Beier-Sexton, Magda; Rahman, M. Sayeedur; Azad, Abdu F.; Gillespie, Joseph J. Publication Date 2018-06 Keywords Metabolic Networks and Pathways; Rickettsia--genetics; Rickettsia--metabolism Download date 01/10/2021 05:45:11 Link to Item http://hdl.handle.net/10713/16003 Wholly Rickettsia! Metabolic Profile of the Quintessential Bacterial Parasite of Eukaryotic Cells Timothy Driscoll a a b b b b b b b Timothy P. Driscoll , Victoria I. Verhoeve , Mark L. Guillotte , Stephanie S. Lehman , Sherri A. Rennoll , Magda Beier-Sexton , M. Sayeedur Rahman , Abdu F. Azad , and Joseph J. Gillespie [email protected] Department of Biology, West Virginia University, Morgantown, WV 26505, USAa; Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD 21201, USAb @driscollMML (twitter, fb, reddit, github) BACKGROUND RESULTS Figure 2. Synopsis of known and predicted metabolites imported from Figure 3. Fatty acids and glycerophospholipids are synthesized Figure 5A. Cell envelope glycoconjugates are synthesized from Bacteria in the genus Rickettsia (Rickettsiaceae; Alphaproteobacteria) Our metabolic reconstruction (Fig. 1) identified 51 host metabolites (including 21 the eukaryotic cytoplasm by rickettsiae. from host precursors dephospho-CoA and biotin, DHAP and G3P. imported host sugars D-ribose 5-P, UDP-glucose, and NAG-1-P. known to be imported Dephospho-CoA Biotin = ATP = SAM N-acetylglucosamine-1-P are obligate intracellular microbes with fragmented genomes and Dihydroxy GpsA sn-Glycerol D-ribose 5-P D-Ribulose 5-P CMP-Kdo previously characterized) required to compensate for the patchwork Rickettsia -acetone = acetyl-CoA = [4Fe-4S] predicted to be imported Rickettsia Imported Metabolite Exact Mass of Metabolite (KDa) 3-phosphate RpiB GlmU CoaE PccA BCCP BirA phosphate = NADPH = CTP diminished metabolic capabilities. They are found widely distributed metabolic network (Fig. 2). Fatty acid and glycerphospholipid synthesis initiate from 0 1 2 3 4 5 6 7 8 = NADH = G3P A V = UTP = L-Serine Flavin adenine dinucleotide ? UDP-glucose UDP-N-acetyl-alpha-D-glucosamine among vertebrate hosts and include several serious human pathogens. host precursors (Fig. 3), and the import of both isoprenes and terpenoids is required for Coenzyme A (CoA) Biotin- Dephosphocoenzyme A PccABCCP (Host derived?) Ugd FnlA WecB WecA LpxA MurA Acyl carrier Lysophosphatidic Nicotinamide adenine dinucleotide AcpS protein (AcpP) acid (LPA) Phosphatidylcholine UDP-glucuronate UDP-alpha-D UDP-N-acetyl-D N-acetyl-alpha-D-glucos- UDP-3-O-(3- UDP-NAG- Although the metabolic intractability of the rickettsiae has been studied the synthesis of ubiquinone and the lipid carrier of Lipid I and O-antigen (data not SAH - PccA PC Flavin mononucleotide HCO 3 -galactose -mannosamine aminyl-diphospho-ditrans, hydroxytetradecanoyl) enolpyruvate 5,6,7,8-Tetrahydrofolate Holo-ACP PlsC choline octacis-undecaprenol -N-acetylglucosamine for decades, few details are known about how rickettsiae acquire the shown). Unlike bacterial symbionts of arthropods, rickettsiae lack the capability for de EamA A Arthropods Thiamine diphosphate PccB Carboxy- Pld Malonyl-CoA biotin- Pld V Vertebrates S-Adenosyl-L-methionine FabD Phosphatidic acid Lipid I metabolites required for their survival. In this study, we integrate recent novo synthesis of B vitamins or most other cofactors (Fig. 4); consequently, we stress PccABCCP Essential metabolite Kdo 2 -lipid A extension; O-antigen polymerization O-antigen carrier Lipid A (Peptidoglycan) Glutathione Pld de novo synthesis Tlc5 Hexadecanoyl-ACP Pyridoxine phosphate Malonyl-ACP CdsA genomic data with historical analyses to construct a comprehensive, the use of “endoparasite” over “endosymbiont” for non-pathogenic rickettsiae. Cell Synthesized from BioY (cycle 7 product) ethanolamine essential metabolite Biotin ADP FabH (cycles 2-8) Phosphatidyl- genus-level metabolic and transport network for the rickettsiae. envelope glycoconjugates are synthesized from three imported host sugars (Fig. 5A), Both 4-hydroxybenzoate CDP-diacylglycerol ethanolamine Figure 5B. Rickettsiae possess a complete TCA cycle, but require a Variable Guanosine 5'-triphosphate (Host derived?) Tlc1 β-Ketoacyl-ACP Acyl-ACP with a range of additional host-acquired metabolites fueling the TCA cycle (Fig. 5B). Six Adenosine 5'-triphosphate FabF range of host-acquired cofactors to fuel it (ATP, CoA, Glx, etc). Uridine 5'-triphosphate PgsA PssA FabG Composition Cytidine 5'-triphosphate FabI Peptidoglycan, biosynthetic pathways contain pathway holes (Fig. 6A; see METHODS), and similar Octanoyl-ACP Phosphoenol- FabZ PE = 60-70% CMP-Kdo (LPS) pyruvate (PEP) Guanosine 5'-diphosphate Tlc4 β-hydroxyacyl-ACP β-enoyl-ACP (cycle 3 product) Phosphatidyl- Phosphatidyl- N-Succinyl-LL-2,6- N-Succinyl-2-L-amino PG = ~20% diaminopimelate -6-oxoheptanedioate patterns in taxonomically diverse bacteria (Fig. 6B) suggest atypical chemistry or Guanosine 5'-monophosphate glycerophosphate L-serine ? Adenosine 5'-monophosphate LipB PS PC = ~15% PpdK DapE Glycero- PdhA CONCLUSIONS utilization of novel metabolites. A paucity of characterized and predicted transporters Uridine 5'-monophosphate PS = trace Suc-CoA DapD phospholipids PdhB PgpA Psd Pyruvate Known to be (2Z,6Z)-Farnesyl diphosphate Protein N6- CL = trace Tlc2, Tlc3 LL-2,6-Diaminopimelate 2,3,4,5-Tetra- Collectively, our work provides a detailed metabolic profile of rickettsial (see Fig. 2) emphasizes the gap in our knowledge concerning how rickettsiae imported Isopentenyl diphosphate (3R)-3-Hydroxy- (3R)-3-Hydroxy- (octanoyl)lysine 2-Hydroxy- tetradecanoyl-ACP hexadecanoyl-ACP Phosphatidyl- Phosphatidyl- hydrodipicolinate TdcB ethyl-ThPP Dimethylallyl diphosphate glycerol ethanolamine ThDP (cycle 6 product) (cycle 7 product) LipA DapF parasites and highlights aspects of reductive genome evolution and import host metabolites, many of which are large and not known to be transported by COG2984 PHB UDP-glucose PG PE L-Serine COG4120 DapB or PdhA Lipo- N-Acetyl-a-D-glucosamine 1-phosphate transacylation addiction to host cell metabolites. We show that NAG-1-P meso-2,6-Diaminopimelate PdhB amide-E the core biosynthesis any described bacteria. COG1101 Protein N6- ? reactions; PhbC D-Ribose 5-phosphate Pi HTPA (lipoyl)lysine Aas phospholipase Phosphorylcholine GlmU_N A1 degradation GlyA hubs in Rickettsia have been supplanted by elaborate thievery of / sn-Glycerol 3-phosphate GlpT Cardiolipin 2-Acyl-sn- DapA (R)-3-hydroxy- / Peptidoglycan butyryl-CoA S-Acetyl- UDP-NAG CL glycero-3- dihydro- Dihydroxyacetone phosphate + Lpd host metabolites to complement a patchwork metabolic network. We ACKNOWLEDGEMENTS H E2 subunits of phospho- L-Aspartate 4-semialdehyde lipoamide-E (S)-Malate SLC5sbd 2-oxoglutarate (Host derived?) ethanolamine L-Glycine LpxA and pyruvate OXPHOS PhbB / Pyruvate YfdV-1, Dihydrolipo- also reveal a startling degree of functional conservation across all 84 Supported with funds from National Institute of Health/National Institute of Allergy and + dehydrogenase YfdV-2 Na ? known to be imported predicted to be imported Asd Fatty Acids amide-E Biosynthetic pathways Acetate Acetoacetyl-CoA UDP-3-O-(3-hydroxy / tetradecanoyl)-N- PdhC sequenced Rickettsia genomes regardless of host or pathogenicity. Infectious Diseases grants (R01AI017828, R01AI126853, R21AI26108, T32AI095190, L-Tryptophan 4-Phospho-L-aspartate MaeB Cofactors/cosubstrates acetylglucosamine Hexadecanoyl-ACP Heme A L-Tyrosine (cycle 7 product) PhbA Ribonucleotides Cationic aa transporters LPS and T32AI007540. We are grateful to Dr. Lucy Weinert (University of Cambridge) for L-Arginine + + Acetyl-CoA Finally, we identify areas of future research aimed at understanding the H H LpxC Octadecanoyl-ACP CtaA LysC Terpenoid backbone L-Phenylalanine UDP- (cycle 8 product) 2-O-(2-hydroxy / sharing the unpublished genomes of Rickettsia species isolated from the ladybird beetle Cell envelope glycoconjugates Heme O AcnA Citrate Oxaloacetate host/parasite interface and elucidating the chemical dependency that L-Histidine AdiC PotE hexadecanoic), RpiB, KdsD, KdsA, KdsB or UDP-3-O-(3-hydroxy GltA Mdh Glycerophospholipids L-Methionine tetradecanoyl)-D- 3-O-(3-hydroxy Kdo2 Lauroyl-Kdo2 Kdo 2 - SucA glucosamine tetradecanoic)- -lipid IV(A) -lipid IV(A) lipid A defines the Rickettsia obligate intracellular lifestyle. Adalia bipunctata and the parasitic ciliate Ichthyophthirius multifiliis. Pyr/Ac-CoA-TCA cycle L-Glutamate LpxD D-glucosamine LpxI, LpxB, LpxK, WaaA LpxL LpxJ CtaB 3-Carboxy- Isocitrate (S)-Malate Proteogenic amino acids L-Lysine Glutamate Heme B 1-hydroxy AspC L-Glutamine propyl-ThPP GlnHPQ Aspartate GlnA Transporters L-Aspartate FumC HemH ThDP Icd / ABC MFS L-Asparagine GltP Lipo- Glutamine L-Leucine Protoporphyrin SucA amide-E GdhX AAA VUT + 2-Oxoglutarate Fumarate Figure 1. Rickettsia metabolic network reconstruction highlights reductive genome evolution and an addiction to host cell metabolites. L-Isoleucine H Figure 4. Rickettsiae lack the capacity for de novo folate synthesis, / / AEC DMT L-Cysteine COG2984 HemJ SdhD S-Succinyl- / / / L-Threonine COG4120 but may use FolE to initiate queuosine synthesis from imported GTP. Succinyl-CoA Succinate SdhC APC SSS Protoporphyrinogen IX dihydro- Lpd FAD, FMN L-Valine
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